286 PROCEEDINGS OF THE AMERICAN ACADEMY. 



ologists, gave me complete confidence in the reliability of this reagent 

 for the material in hand. The results in this case wei-e in the highest 

 degree satisfactory. 



After the specimens had been entirely freed from acid, drawings of 

 them as opaque objects seen against a black background were made 

 with the aid of an Abbe camera lucida ; they were then returned 

 to 70% alcohol, and from this transferred to Kleineubers's alcoholic 

 hcEmatoxylin (70%) diluted with twice its volume of his solution ot 

 calcium chloride and alum. Here they remained for a few hours, the 

 time varying according to the size of the embryos, and were then 

 transferred to a 0.1% solution of hydrochloric acid in 70% alcohol, 

 the process of decolorizing being carefully watched under the micro- 

 scope until the object had attained the proper color. It was then 

 placed in 70% alcohol containing a slight trace of ammonia, which 

 made the stain permanent by neutralizing the acid. I have found that 

 washing simply in neutral alcohol, though it be never so carefully done, 

 will not always prevent an ultimate fading of this stain ; the addition 

 of ammonia is an absolute essential if one wishes to be perfectly sure 

 that the sections will not fade. Objects which I have treated thus have 

 always preserved their color. In my work on the pig I have not as 

 yet used carmine stains. On the embryos of both the rat and the 

 mouse I have obtained very brilliant results, both with borax carmine 

 and with hydrochloric carmine, but, so far as I could see, they had no 

 advantage over ha3matoxylin. The last is valuable because of its 

 high alcoholic grade, and because, when properly decolorized, it be- 

 comes a most highly differential stain for these embryonic tissues. 

 Small semi-transparent objects, like the embryos of the pig at this 

 stage of development, can readily be decolorized in toto, for the extent 

 of the decolorizing can be determined by the aid of the microscope ; 

 but opaque objects, like the uterus of the mouse, have to be decolorized 

 largely after sectioning. 



When the embryos had been stained and decolorized, they were 

 cleared in chloroform, embedded in paraffine, and cut on the Minot- 

 Zimmerman microtome into sections 10^ thick. They were then 

 spread on the surface of distilled water, which rested on a thin film of 

 albumen affixative covering the slide. The slide was gently warmed 

 in an alcohol flame, until the sections became perfectly smooth. 

 Finally the water was removed, the paraffine dissolved out in xylol, 

 and the sections mounted in Canada balsam. 



I have given this rather extended account of my technique in order 

 to show that there has been no lack of care in this direction which 



